Doxorubicin-Induced Modulation of TGF-β Signaling Cascade in Mouse Fibroblasts: Insights into Cardiotoxicity Mechanisms

Doxorubicin (DOX)-induced cardiotoxicity has been widely observed, yet the specific impact on cardiac fibroblasts is not fully understood. Additionally, the modulation of the transforming growth factor beta (TGF-β) signaling pathway by DOX remains to be fully elucidated. This study investigated DOX’s ability to modulate the expression of genes and proteins involved in the TGF-β signaling cascade in mouse fibroblasts from two sources by assessing the impact of DOX treatment on TGF-β inducible expression of pivotal genes and proteins within fibroblasts. Mouse embryonic fibroblasts (NIH3T3) and mouse primary cardiac fibroblasts (CFs) were treated with DOX in the presence of TGF-β1 to assess changes in protein levels by western blot and changes in mRNA levels by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Our results revealed a dose-dependent reduction in cellular communication network factor 2 (CCN2) protein levels upon DOX treatment in both NIH3T3 and CFs. Moreover, we observed that DOX inhibited the TGF-β1 induced expression of BMP1 in NIH3T3 cells, while BMP1 levels remained high in CFs, and that TGF-β1 induces the phosphorylation of SMAD2 in both NIH3T3 cells and CFs. While DOX treatment diminished the extent of phosphorylation, the reduction did not reach statistical significance. DOX also inhibited the TGF-β1 induced expression of COL1 in NIH3T3 cells and CFs. Finally, DOX inhibited the TGF-β1 induced expression of Atf4 and increased the expression of Cdkn1a, Id1, Id2, Runx1, Tgfb1, Inhba, Thbs1, Bmp1, and Stat1 in NIH3T3 cells but not CFs, indicating the potential for cell-specific responses to DOX and its modulation of the TGF-β signaling pathway. Understanding the underlying mechanisms of the ability of DOX to modulate gene expression and signaling pathways in fibroblasts holds promise for future development of targeted therapeutic strategies to mitigate DOX-induced cardiotoxicity specifically affecting CFs.


Introduction
Chemotherapy has improved cancer survival rates, however, side effects to chemotherapeutic drugs remains a concern. An increased risk of cardiovascular complications and cardiotoxicity is attributed to anthracyclines [1][2][3][4] . Anthracyclines are a class of naturally occurring antibiotics produced by Streptomyces bacteria that exhibit broad anticancer e cacy. One of the most widely used anthracyclines is DOX, which was approved by the FDA in 1974 for the treatment of cancers, including hematopoietic cancers such as leukemias and lymphomas, and metastatic solid tumors such as breast, gastric, neuroblastoma, lung, ovarian, and bladder cancers in both adult and pediatric patients 5 . Despite more than ve decades of research, the clinical use of DOX is limited by its dose-dependent cardiotoxic side effects, which remain a serious risk to patients.
Echocardiography and cardiac magnetic resonance imaging are standard methods used to monitor the left ventricle ejection fraction (LVEF) to assess the progression of cardiotoxicity induced by DOX 6-10 .
DOX-induced cardiotoxicity is characterized by a gradual decline in LVEF which, if not managed, can lead to heart failure. A signi cant reduction in LVEF is de ned as a decrease of more than 10% from baseline to a value below the lower limit of normal (usually less than 50-55%) [10][11][12] . The risk of developing cardiotoxicity increases with the cumulative dose of DOX 13 . The reported risk of DOX-induced cardiotoxicity by cumulative dose is 3-5% for 400 mg/m 2 , 7-26% for 550 mg/m 2 , and 18-48% for 700 mg/m 2 14-18 . Moreover, age is a signi cant factor that contributes to the susceptibility to cardiotoxicity in DOX-treated patients. Patients under ve years old or over sixty-ve years old are more vulnerable to developing cardiotoxicity 14,19 . It is likely that these age groups are more prone to cardiotoxicity due to age-related differences in the cardiovascular system which also increase the risk of developing heart failure.
DOX exerts its antitumor activity by intercalating into DNA, which inhibits the binding of topoisomerase IIβ and the unwinding of supercoiled DNA [20][21][22][23] . This inhibition leads to a blockage of DNA synthesis and the eventual induction of double-stranded DNA breaks, triggering programmed cell death in cells 24,25 .
Despite its therapeutic bene ts, DOX is associated with off-target effects, which include the production of reactive oxygen species (ROS). The precise mechanisms underlying DOX-induced ROS overproduction are not fully understood, however, studies suggest that mitochondrial dysfunction 26, 27 , calcium homeostasis 28, 29 , iron chelation 30,31 , and in ammatory responses [32][33][34][35] are implicated in ROS generation caused by DOX. Although antioxidants have been used in combination with DOX to decrease the buildup of free radicals, this approach has not proven effective in reducing cardiotoxicity while maintaining the antitumor e cacy of DOX 36 . As a result, the need for alternative therapeutic options to lower the risk of cardiotoxicity from DOX remains.
DOX-induced cardiotoxicity research has primarily focused on cardiomyocytes. The impact of DOX on CFs remains underexplored. The myocardium is a complex tissue comprising multiple cell types, including CFs, which are the most abundant cell type in the left ventricle of the heart 37 . CFs play a crucial role in regulating the expression and accumulation of extracellular matrix (ECM) components such as collagens, bronectin, matrix metalloproteinases (MMPs), and endogenous protease inhibitors known as tissue inhibitors of metalloproteinases (TIMPs) 38-42 . Despite their importance, the study of CFs in DOXinduced cardiotoxicity has been limited compared to cardiomyocytes. Therefore, there is a need to assess the contribution of CFs to DOX-induced cardiotoxicity and bridge the knowledge gap in this area.
Transforming growth factor β (TGF-β) is a pivotal cytokine/growth factor that has been shown to stimulate broblasts 43,44 . The TGF-β signaling pathway has been implicated in brosis in the myocardium, which contributes to cardiac dysfunction [45][46][47][48][49] . In this study, we investigated the effects of DOX on TGF-β signaling cascade in mouse broblasts from two sources to better understand the mechanisms of its cardiotoxicity, which in turn may facilitate the identi cation of potential therapeutic targets to alleviate this serious adverse effect of DOX.

Effect of DOX on broblast viability
Cell viability of NIH3T3 and CFs was evaluated using an alamarBlue assay. Cells were treated with 0-50 µM DOX for 24 hours. A signi cant decrease in cell viability was observed at 2.5 µM DOX and above (Fig. 1). Thus, sublethal concentrations of DOX (≤ 1.0 µM) were used in the subsequent experiments. DOX treatment Inhibited TGF-β induction of CCN2 protein level in a dose-dependent manner In this study, we evaluated the expression of cellular communication network factor 2 (CCN2) protein in NIH3T3 and CFs treated with DOX (Fig. 2). A dose-dependent downregulation of CCN2 protein expression was observed in both cell lines treated for 24 hours with DOX and TGF-β1. These results suggest that DOX modulates the expression of CCN2 in broblasts and that DOX may play a role in ECM remodeling.

DOX treatment inhibited the TGF-β-induced expression of BMP1 in NIH3T3 broblasts
In this study, we examined the impact of DOX on the expression of BMP1 protein in NIH3T3 and CFs using western blot analysis. We observed signi cant changes in BMP1 protein levels in NIH3T3 cells induced by TGF-β1, but not in CFs (Fig. 3).
These ndings indicate that DOX has a cell-speci c effect on BMP1 protein levels in broblasts. Speci cally, the observed changes in BMP1 levels in response to DOX treatment were evident in NIH3T3 cells. In CFs, BMP1 expression levels were higher under all conditions compared to NIH3T3 cells, indicating differences between the NIH3T3 cells and primary CFs. Further investigations are warranted to elucidate the underlying mechanisms and functional implications of these cell-speci c effects on BMP1 expression in the context of TGF-β signaling and DOX treatments.
TGF-β1 induced the phosphorylation of SMAD2 after one hour in both NIH3T3 cells and CFs.
To investigate whether DOX affects the activation of the TGF-β/SMAD2 signaling pathway, we assessed SMAD2 and pSMAD2 in NIH3T3 and CFs treated with TGF-β1 and DOX for one hour 51 . Our results indicated that TGF-β1 stimulation led to a signi cant increase in pSMAD2 in both cell lines, in agreement with previous ndings 52 . DOX treatment diminished the extent of phosphorylation of SMAD2, however the differences did not reach statistical signi cance during the one-hour treatment. Additionally, SMAD2 protein levels decreased slightly in response to DOX compared to control in CFs. This decrease was attenuated by exogenous TGF-β1 (Fig. 4).

DOX treatment inhibited the TGF-β-induced COL1 protein expression in NIH3T3 broblasts and CFs
To evaluate the effects of DOX on COL1 protein expression in NIH3T3 and CFs, we conducted western blot analysis following a 24-hour treatment with DOX and TGF-β1. The ndings demonstrated that TGF-β1 treatments led to an increase in COL1 protein. Additionally, DOX was able to inhibit the TGF-β1 induction of COL1 protein (Fig. 5). These ndings indicate that DOX treatment hinders COL1 production, potentially affecting the deposition of mature collagen within the ECM surrounding cardiac broblasts.
The observed reduction in COL1 protein expression, resulting from DOX treatment indicated that further investigations are warranted to elucidate the underlying mechanisms through which DOX and TGF-β1 interact in CFs. Understanding these interactions could provide valuable insights into the development of strategies for mitigating the adverse effects of DOX on cardiac function and ECM production and remodeling.
DOX inhibited the TGF-β1-induced differential gene expression reduction of Atf4 and promoted differential increased gene expression of Id1, Runx1, Thbs1, Stat1, and Cdkn1a in NIH3T3 Fibroblasts In this study, we examined the effects of DOX on TGF-β1-induction of differential gene expression of negative and positive modulators of the TGF-β/BMP signaling pathway using qRT-PCR. DOX-induced differential expression of several gene in this pathway were observed in NIH3T3 cells, including upregulation of Id1, Id2, Runx1, Tgfb1, Inhba, Thbs1, Bmp1, and Stat1, and downregulation of Atf4 (Fig. 6, Table 1). In contrast, no signi cant gene alterations were observed in CFs under the same conditions ( Supplementary Fig. 1). These ndings indicate that DOX impacts TGF-β and BMP signaling in a cell type-speci c manner.

Discussion
This study provides data on the effects of DOX and TGF-β1 treatment on NIH3T3 and CFs, assessing speci c target proteins of the TGF-β/BMP signaling pathway using western blot and qRT-PCR.
DOX is a widely used antibiotic with broad antitumor e cacy for treating hematopoietic and metastatic solid tumor cancers. However, its cardiotoxic side effect limits its clinical use. DOX-induced cardiotoxicity may manifest within hours of initial treatment and persist up to twenty years post-nal administration, posing a potential lifelong risk for patients treated with DOX [13][14][15][16][17][18] . Despite ve decades of research, there is still no therapeutic option to reduce the risk of developing cardiotoxicity while maintaining its antitumor e cacy. Most research conducted on the matter has focused on cardiomyocytes, the chief cell type within the myocardium responsible for generating contractile force. However, the proper function of cardiomyocytes and the heart relies on other cell types, including CFs, which regulate the cardiac ECM through the TGF-β signaling pathway 53 . The cardiac ECM provides scaffolding and support throughout the heart, facilitates force transmissions for proper pumping, and supplies neighboring cells with an extracellular environment. Therefore, CFs may be a potential therapeutic intervention target for reducing the risk of cardiotoxicity upon DOX treatment.
Cardiac broblasts are a crucial component of the myocardium and play a signi cant role in cardiac function. In CFs, the TGF-β1 signaling pathway regulates various cellular processes like differentiation, migration, proliferation, brosis, and ECM deposition. The TGF-β1 signaling pathway initiates with the binding of its ligand to the cell surface receptor complex, triggering the phosphorylation of SMAD2. Once phosphorylated, pSMAD2 forms a complex with SMAD4 and translocates into the nucleus, where it acts as a transcription factor to promote the expression of TGF-β target genes [54][55][56] . In broblasts, SMAD2/pSMAD2 plays a crucial role in regulating the expression of various ECM genes, including collagens, CCN2, MMP2, and MMP9, which are involved in tissue remodeling processes following injury 45,57,58 . Dysregulation of SMAD2/pSMAD2 has been implicated in several pathological conditions, particularly those related to cardiac brosis [59][60][61][62] .
CCN2 is a multifunctional matricellular protein involved in many biological processes, including ECM production, migration, differentiation, angiogenesis, and apoptosis 63-65 . CCN2 has been shown to be a downstream mediator of the TGF-β/SMAD signaling pathway, which play essential roles in the ECM synthesis and remodeling in the heart 66,67 . Activation of these signaling pathways leads to the upregulation of CCN2 expression, which in turn stimulates the production of ECM components and inhibits ECM degradation, ultimately contributing to the maintenance of the cardiac structure and function 68 .
BMP1 is a member of the astacin family of matrix metalloproteinases that play a critical role in the regulation of ECM synthesis and remodeling. In broblasts, BMP1 is involved in the regulation of TGF-β signaling by activating the TGF-β signaling pathway by cleaving the pro-domain of TGF-β precursors, which releases the active TGF-β ligand that then binds to its cell surface receptor complex and activates downstream SMAD proteins 69,70 . BMP1 also cleaves several ECM proteins, including type I procollagen, which matures into the abundant COL1 71,72 .
COL1 represents a major component of the ECM in the heart, contributing to its structural integrity and tensile strength. Moreover, COL1 acts as a crucial scaffold for cardiac cells and actively participates in cardiac remodeling in response to injury or stress. Its presence is vital for the maintenance of cardiac function and the prevention of cardiovascular diseases 73 . The expression of Col1 gene is regulated by the TGF-β signaling pathways, which stimulate the production of type I procollagen, subsequently undergoing maturation and deposition within the extracellular space 74 .
The cardiac ECM plays a crucial role in maintaining cardiac homeostasis by continuously interacting with cellular elements, providing structural support, and coordinating cellular responses to ensure proper myocardial function. ECM regulation is achieved through the highly controlled synthesis of matrix proteins and proteolytic processing facilitated by MMPs and TIMPs. The dysregulation of the cardiac ECM can impair cardiac function and limit signaling between cells which can modulate cell behavior and lead to pathological changes in the heart.
This study focused on investigating the effect of DOX treatment on targets of the TGF-β signaling pathway in two mouse broblast cell lines. One of the key ndings of this study is the dose-dependent downregulation of cellular CCN2 protein expression in both NIH3T3 broblasts and CFs upon DOX treatment. CCN2 plays a crucial role in ECM synthesis and remodeling in the heart. Activation of TGF-β signaling leads to the upregulation of CCN2 expression, which stimulates ECM production and inhibits ECM degradation. The background expression of CCN2 is usually very low, therefore, cells were stimulated with TGF-β1 (5 ng/mL) to induce CCN2 expression. The observed downregulation of CCN2 protein expression suggests that DOX treatment can modulate the expression of CCN2, potentially impacting ECM remodeling processes.
The TGF-β/SMAD signaling pathway is critical in regulating ECM gene expression and tissue remodeling. We evaluated SMAD2 protein expression and phosphorylation levels in response to DOX treatments. The treatment of TGF-β1 signi cantly increased the pSMAD2 expression in both NIH3T3 and CFs compared to treatment groups without the exogenous TGF-β1. A decrease in SMAD2 protein expression was observed in DOX-treated CFs compared to the control group. However, the response did not reach statistical signi cance in the TGF-β1 treated groups.
BMP1 is an enzyme involved in TGF-β signaling and ECM regulation. BMP1 is known to activate the TGFβ pathway and cleave ECM proteins such as type I procollagen. Our results showed a signi cant change in BMP1 protein levels in response to DOX treatment in NIH3T3 cells stimulated with TGF-β1. These ndings suggest a cell-speci c response to DOX treatment and indicate the need for further investigation to understand the underlying mechanisms and functional implications of these differential effects. Furthermore, while protein levels were decreased by DOX treatment, mRNA levels were slightly increased, indicating that transcription may be increased but translation is not. COL1 protein is a major structural component of the cardiac ECM. COL1 provides structural integrity to the heart and plays a crucial role in cardiac remodeling. The TGF-β signaling pathway regulates COL1 expression, and our results demonstrated that DOX treatment, signi cantly reduced the TGF-β1-induced COL1 protein increase compared to TGF-β1 treatment alone. These ndings suggest that DOX treatment impaired the production of collagen destined for the ECM, potentially affecting the cardiac remodeling processes and resulting in cardiac tissue integrity.
Moreover, this study examined the effects of DOX on gene transcription in the TGF-β and BMP signaling pathways. Signi cant alterations in gene expression for 10 genes were observed in NIH3T3 cells treated with DOX and TGF-β1, however, no differential gene expression was observed in CFs under the same conditions.
Speci cally, we observed that Activating Transcription Factor 4 (Atf4), a gene that regulates various cellular processes, was signi cantly downregulated in NIH3T3 cells treated with TGF-β1 and DOX compared to TGF-β1 treatment alone. Prior reports have indicated a positive correlation between ATF4 protein levels and the expression of COL1, as evidenced by signi cant reductions in type 1 collagen biosynthesis in ATF4 knockout mice 75 . These ndings suggest a potential association between decreased expression of the Atf4 gene and reduced COL1 proteins expression. Further evidence is required to elucidate whether treatments of DOX affects ATF4 and the synthesis of ECM components in broblasts.
On the other hand, the inhibitor of DNA binding 1 (Id1), which is a transcriptional repressor that regulate genes involved in cellular processes such as proliferation, differentiation, and apoptosis, was signi cantly upregulated in the presence of DOX. Id1 is an inhibitor of the TGF-β-induced collagen expression in human dermal broblasts 76 . Additionally, in stem-cell derived endothelial cells, TGF-β inhibition was found to be Id1 dependent 77 . This would suggest that DOX negatively correlates with TGF-β signaling increasing Id1 expression and reducing collagen deposition.
We also found that Runt-related Transcription Factor 1 (Runx1) also known as AML1/ETO was upregulated. RUNX1 has been demonstrated in various cancer studies to inactivate the TGF-β1/SMAD signaling pathway 78 . This pathway is responsible for regulating the expression of various ECM components such as collagens, bronectins, MMPs and TIMPs.
Interestingly, there was an upregulation of Tgfb1, transforming growth factor beta 1, mRNA. Additionally, Thrombospondin 1 (Thbs1) mRNA was also found to be elevated in the presence of DOX + TGF-β1 compared to TGF-β1 treatment alone. Thbs1 promotes Tgfb1 expression in broblasts in vitro 79 . This suggests that DOX-induced Thbs1 mRNA expression explaining the increase observed in Tgfb1 mRNA expression. Further research is required to elucidate the effects of DOX on Tgfb1 and Thbs1 mRNA in broblast.
Signal Transducer and Activator of Transcription 1 (Stat1), a downstream target of the TGF-β signaling pathway, was upregulated in NIH3T3 cells upon treatment with DOX + TGF-β1 compared to TGF-β1 treatment alone. Stat1 has been observed to inhibit the myo broblast phenotype in cells of broblastic origin in vivo 80 . This would suggest that DOX prevents the transformation of broblasts to myo broblasts.
Lastly, cyclin dependent kinase inhibitor 1A (Cdkn1a) (also known as p21), is a senescence marker for oxidative stress that induces G2 arrest in the cell cycle in human broblasts 81-84 .
The downregulation of Atf4 and upregulation of Id1, Runx1, and Stat1 in NIH3T3 cells suggest that DOX treatments may have an inhibitory effect on ECM deposition. These ndings imply a potential reduction in the synthesis and deposition of ECM components. However, it is important to note that Cdkn1a was also upregulated in NIH3T3 cells. Cdkn1a is known to play a role in the transformation of broblasts into myo broblasts. This nding appears contradictory to the previously mentioned Atf4, Id1, Runx1, and Stat1 in reducing ECM deposition as myo broblasts are pro-brotic in nature and promote the biosynthesis and deposition of ECM. It is possible that the upregulation of Cdkn1a mRNA observed in this study was a result of oxidative stress caused by the 24-hour treatment of DOX, rather than a direct response to broblast transformation into myo broblasts. Further investigation is required to fully elucidate the effects of DOX on these genes and clarify the underlying mechanisms.
In summary, this study provides valuable insights into the effects of DOX on broblast function, ECM remodeling, and the TGF-β signaling pathway involved in cardiac tissue homeostasis. The ndings suggest that DOX treatment can modulate the expression of key proteins involved in TGF-β signaling, ECM synthesis, and remodeling, such as CCN2 and COL1. The differential effects observed in BMP1 protein levels highlight the cell-speci c responses to DOX treatment. A small, but detectable increase in total SMAD2 protein was observed when comparing DOX treatment to DOX plus TGF-β1, and this may suggest that TGF-β1 as a co-treatment with DOX may lessen the side effects and may be a potential avenue for future investigation of therapeutic intervention. Further investigations are warranted to elucidate the underlying mechanisms and functional implications of these observations, which could contribute to the development of strategies for mitigating DOX-induced cardiotoxicity and preserving cardiac function in clinical settings.

Materials and Methods
Cell Culture Thermo Fisher Scienti c; 11965092) supplemented with 10% (v/v) fetal bovine serum (Atlanta Biologicals; S22660), 5 mL of 10 units/L penicillin, and 5 mL 10 mg/mL streptomycin (Thermo Fisher Scienti c; 15140122). Prior to seeding CFs, all culture vessels were pre-coated with a gelatin-based coating solution (Cell Biologics; 6950) for one hour. Both cell lines were maintained under standard conditions of 5% CO 2 at 37°C and 95% humidity.

Assessment of Cell Viability of Fibroblasts treated with DOX
Cell viability of broblast cell lines treated with DOX were assessed by alamarBlue assay (Thermo Fisher Scienti c; A50100) at a seeding density of 1 × 10 4 cells per well (100 µL) in 96-well at bottom plates. The cells were treated with a series of DOX concentrations ranging from 0 to 50 µM for 24 hours. After 21 hours, 10 µL of 10X alamarBlue was added to each well, and incubated for three hours. Fluorescence conversion was measured using a Synergy H1 microplate reader (Biotek) with 560 nm excitation and 590 nm emission.
Protein expression analysis by western blot NIH3T3 and CFs were seeded at a density of 2 × 10 5 cells per well (2 mL) in 6-well plates one day prior to their treatments. Treatments were administered for 24 hours, except for the SMAD2 phosphorylation study, which lasted for one hour. Cells were treated with DOX (1 µM; 0.25, 1, and 2.5 µM for CCN2 study) and 5 ng/mL TGF-β1 or the respective vehicle control, DMSO and 4 mM HCl, respectively. ECM synthesis was promoted with 100 µg/mL ascorbic acid during treatments 50

Statistical Analysis
The values were presented as mean ± SE. Differences between the two groups were evaluated using the Student t-test. Differences among multiple groups were evaluated using a two-way analysis of variance (ANOVA) with Dunnett's test. The level of signi cance was selected to be P < 0.05. R statistics software was used to perform the statistical analysis.
Declarations funding from the National Science Foundation, Grants #0619793 and #0923535; the M. J. Murdock Charitable Trust; Lori and Duane Stueckle, and the Idaho State Board of Education.
Author Contributions: X. P. and J. T. O. conceived the experiments. C. P. and P. L. conducted the experiments. All authors analyzed the results and reviewed the manuscript.
Data availability statement: The authors con rm that the data supporting the ndings of this study are available within the article and its supplementary materials.
Con icts of Interest: The authors declare no con ict of interest.
Cell viability remains high at DOX concentrations less than 1 µM. Cell viability analysis was performed using the alamarBlue assay on NIH3T3 cells and CFs exposed to speci c concentrations of DOX for 24 hours. Error bars represent ± standard error represented a sample size of n=8 for NIH3T3 and n=6 for CFs. µg/mL ascorbic acid for 24 hours. Values represent mean ± standard error (n=4). Statistical analysis performed with two-way ANOVA. * P-value < 0.05 is compared between the control group and the treatment group stimulated with TGF-β1 without DOX, and # P-value < 0.05 are treatments compared to the treatment group stimulated with TGF-β1 without DOX.

Figure 3
DOX inhibited the TGF-β1 induced expression of BMP-1 in NIH3T3 cells, while BMP-1 levels remained high in CFs. The effect of DOX on BMP1 protein expression in NIH3T3 cells (A) and cardiac broblasts (CFs) (B), with and without TGF-β1 stimulation was assessed. Cells were treated with 1 µM DOX and 100 μg/mL ascorbic acid for 24 hours, and protein extracts were collected for western blot analysis.
Expression levels of BMP1 and the housekeeping protein β-actin were determined, and densitometry was performed for BMP1, normalized to β-actin. Error bars represent ± standard error (n=4). Statistical analysis was completed with Student's t-test assuming unequal variance. # P-value < 0.05.

Figure 4
TGF-β1 induced the phosphorylation of SMAD2 in both NIH3T3 cells and CFs. DOX treatment diminished the extent of phosphorylation; however, the reduction did not reach statistical signi cance. The effect of DOX on SMAD2 and phosphorylated SMAD2 (pSMAD2) in NIH3T3 cells (a) and CFs (B) was assessed.
Cells were treated with 1 mM DOX and 100 mg/mL ascorbic acid and stimulated with or without TGF-β1for one hour. Densitometry was performed on SMAD2, pSMAD2, and normalized to housekeeping protein β-actin. Values represent mean ± standard error (n=4). * P-value < 0.05 when compared to treatments without DOX and # P-value < 0.05 compared to treatments with DOX. & P-value < 0.05 are treatments with and without DOX without any TGF-β1 stimulation. Cells were treated with 1 µM DOX and 100 μg/mL ascorbic acid for 24 hours, with or without 10 ng TGF-β1 stimulation, after which protein extracts were collected and subjected to western blotting to determine the expression levels of COL1 and the housekeeping protein β-actin. Densitometry was performed for COL1 and normalized to β-actin. Values represent mean ± standard error (n=4). P-values were determined using Student t-test assuming unequal variance * P-value < 0.05 control compared to treatment stimulated with TGF-β1 without DOX, and # P-value < 0.05 compared to both treatment groups treated with TGF-β1.